MRM/SRM assay for death receptor 5 protein

ABSTRACT

Specific peptides, and derived ionization characteristics of those peptides from Death Receptor 5 (DR5) protein are provided that are particularly advantageous for quantifying the DR5 protein directly in biological samples that have been fixed in formalin by the method of Selected Reaction Monitoring/Multiple Reaction Monitoring (SRM/MRM) mass spectrometry. Such biological samples are chemically preserved and fixed wherein the biological sample is selected from tissues and cells treated with formaldehyde containing agents/fixatives including formalin-fixed tissue/cells, formalin-fixed/paraffin embedded (FFPE) tissue/cells, FFPE tissue blocks and cells from those blocks, and tissue culture cells that have been formalin fixed and or paraffin embedded. A protein sample is prepared from a biological sample using the Liquid Tissue™ reagents and protocol, and the DR5 protein are quantitated in the Liquid Tissue™ sample by the method of SRM/MRM mass spectrometry by quantitating in the protein sample at least one or more of the peptides described for one or more of the DR5 protein. These peptides can be quantitated if they reside in a modified or in an unmodified form. An example of a modified form of a DR5 peptide is phosphorylation of a tyrosine, threonine, serine, and/or other amino acid residues within the peptide sequence.

This application is a divisional application of U.S. patent applicationSer. No. 14/223,464, filed Mar. 24, 2014, now U.S. Pat. No. 9,442,119,which is a continuation of International Application No. PCT/US12/56961,filed Sep. 24, 2012, which claims the benefit of U.S. ProvisionalApplication No. 61/538,096, filed Sep. 22, 2011, entitled “MRM/SRM Assayfor Death Receptor 5 Protein,” the contents of each of which are herebyincorporated by reference in their entireties. This application alsocontains a sequence listing submitted electronically via EFS-web, whichserves as both the paper copy and the computer readable form (CRF) andconsists of a file entitled “001152 8025 US02 SEQ LISTING”, which wascreated on Jul. 14, 2016, which is 912 bytes in size, and which is alsoincorporated by reference in its entirety.

INTRODUCTION

Specific peptides derived from subsequences of the Death Receptor 5(DR5) protein are provided. The peptide sequences andfragmentation/transition ions for each peptide are particularly usefulin a mass spectrometry-based Selected Reaction Monitoring (SRM) assay,which can also be referred to as a Multiple Reaction Monitoring (MRM)assay. Such assays are referred to herein as SRM/MRM. The use ofpeptides for quantitative SRM/MRM analysis of the DR5 proteins isdescribed.

This SRM/MRM assay can be used to measure relative or absolutequantitative levels of one or more of the specific peptides from the DR5protein. This provides a means of measuring the amount of the DR5protein in a given protein preparation obtained from a biological sampleby mass spectrometry.

More specifically, the SRM/MRM assay can measure these peptides directlyin complex protein lysate samples prepared from cells procured frompatient tissue samples, such as formalin fixed cancer patient tissue.Methods of preparing protein samples from formalin-fixed tissue aredescribed in U.S. Pat. No. 7,473,532, the contents of which are herebyincorporated by reference in their entirety. The methods described inU.S. Pat. No. 7,473,532 may conveniently be carried out using LiquidTissue™ reagents and protocol available from OncoPlexDx (formerlyExpression Pathology Inc., Rockville, Md.).

Results from the SRM/MRM assay, where the proteins are analyzedindividually or simultaneously, can be used to correlate accurate andprecise quantitative amounts of these proteins within specific tissuesamples (e.g., cancer tissue sample) of the patient or subject from whomthe tissue (biological sample) was collected and preserved. This notonly provides diagnostic information about the cancer, but also permitsa physician or other medical professional to determine appropriatetherapy for the cancer patient. Such an assay that providesdiagnostically and therapeutically important information about levels ofprotein expression in a diseased tissue or other patient sample istermed a “companion diagnostic” assay. For example, such an assay can bedesigned to diagnose the stage or degree of a cancer and determine atherapeutic agent to which a patient is most likely to respond.

SUMMARY

The assays described herein measure relative or absolute levels ofspecific unmodified peptides from the DR5 protein and also can measureabsolute or relative levels of specific modified peptides from the DR5protein. Examples of modifications include phosphorylated amino acidresidues (e.g. phosphotyrosine, phosphoserine and phosphothreonine) andglycosylated amino acid residues (e.g. glycosylated asparagine residues)that are present on the peptides.

Relative quantitative levels of the DR5 protein are determined by theSRM/MRM methodology, for example, by comparing SRM/MRM signature peakareas (e.g., signature peak area or integrated fragment ion intensity)of an individual DR5 peptide in different samples.

Alternatively, it is possible to compare multiple SRM/MRM signature peakareas for multiple DR5 signature peptides, where each peptide has itsown specific SRM/MRM signature peak, to determine the relative DR5protein content in one biological sample with the DR5 protein content inone or more additional or different biological samples. In this way, theamount of a particular peptide, or peptides, from the DR5 protein, andtherefore the amount of the DR5 protein, is determined relative to thesame DR5 peptide, or peptides, across 2 or more biological samples underthe same experimental conditions. In addition, relative quantitation canbe determined for a given peptide, or peptides, from the DR5 proteinwithin a single sample by comparing the signature peak area for thatpeptide by SRM/MRM methodology to the signature peak area for anotherand different peptide, or peptides, from a different protein, orproteins, within the same protein preparation from the biologicalsample. In this way, the amount of a particular peptide from the DR5protein, and therefore the amount of the DR5 protein, is determinedrelative one to another within the same sample. These approachesgenerate quantitation of an individual peptide, or peptides, from theDR5 protein to the amount of another peptide, or peptides, betweensamples and within samples wherein the amounts as determined by peakarea are relative one to another, regardless of the absolute weight tovolume or weight to weight amounts of the DR5 peptide in the proteinpreparation from the biological sample. Relative quantitative data aboutindividual signature peak areas between different samples are normalizedto the amount of protein analyzed per sample. Relative quantitation canbe performed across many peptides from multiple proteins and the DR5protein simultaneously in a single sample and/or across many samples togain insight into relative protein amounts, one peptide/protein withrespect to other peptides/proteins.

Absolute quantitative levels of the DR5 protein are determined by, forexample, the SRM/MRM methodology whereby the SRM/MRM signature peak areaof an individual peptide from the DR5 protein in one biological sampleis compared to the SRM/MRM signature peak area of an exogenously added“spiked” internal standard. In one embodiment, the internal standard isa synthetic version of the same exact DR5 peptide that contains one ormore amino acid residues labeled with one or more heavy isotopes.Suitable isotope-labeled internal standards are synthesized so that,when analyzed by mass spectrometry, each standard generates apredictable and consistent SRM/MRM signature peak that is different anddistinct from the native DR5 peptide signature peak and which can beused as a comparator peak. Thus, when the internal standard is spiked ina known amount into a protein preparation from a biological sample andanalyzed by mass spectrometry, the SRM/MRM signature peak area of thenative peptide from the sample can be compared to the SRM/MRM signaturepeak area of the internal standard peptide. This numerical comparisonprovides either the absolute molarity and/or absolute weight of thenative peptide present in the original protein preparation from thebiological sample. Absolute quantitative data for fragment peptides aredisplayed according to the amount of protein analyzed per sample.Absolute quantitation can be performed across many peptides, and thusproteins, simultaneously in a single sample and/or across many samplesto gain insight into absolute protein amounts in individual biologicalsamples and in entire cohorts of individual samples.

The SRM/MRM assay method can be used to aid diagnosis of the stage ofcancer, for example, directly in patient-derived tissue, such asformalin fixed tissue, and to aid in determining which therapeutic agentwould be most advantageous for use in treating that patient. Cancertissue that is removed from a patient either through surgery, such asfor therapeutic removal of partial or entire tumors, or through biopsyprocedures conducted to determine the presence or absence of suspecteddisease, is analyzed to determine whether or not a specific protein, orproteins, and which forms of proteins, are present in that patienttissue. Moreover, the expression level of a protein, or multipleproteins, can be determined and compared to a “normal” or referencelevel found in healthy tissue. Normal or reference levels of proteinsfound in healthy tissue may be derived from, for example, the relevanttissues of one or more individuals that do not have cancer.Alternatively, normal or reference levels may be obtained forindividuals with cancer by analysis of relevant tissues not affected bythe cancer.

Alternatively, normal or reference levels may be obtained forindividuals with cancer by analysis of relevant tissues not affected bythe cancer. Assays of protein levels (e.g., DR5 levels) can also be usedto diagnose the stage of cancer in a patient or subject diagnosed withcancer by employing the DR5 levels. Levels of an individual DR5 peptideis defined as molar amount of the peptide determined by the SRM/MRMassay per total amount of protein lysate analyzed. Information regardingDR5 can thus be used to aid in determining stage or grade of a cancer bycorrelating the level of the DR5 protein (or fragment peptides of theDR5 protein) with levels observed in normal tissues.

Assays of protein levels (e.g., DR5 levels) can also be used to diagnosethe stage of cancer in a patient or subject diagnosed with cancer byemploying the DR5 levels. Levels or amounts of proteins or peptides canbe defined as the quantity expressed in moles, mass or weight of aprotein or peptide determined by the SRM/MRM assay. The level or amountmay be normalized to total the level or amount of protein or anothercomponent in the lysate analyzed (e.g., expressed inmicromoles/microgram of protein or micrograms/microgram of protein). Inaddition, the level or amount of a protein or peptide may be determinedon volume basis, expressed, for example, in micromolar ornanograms/microliter. The level or amount of protein or peptide asdetermined by the SRM/MRM assay can also be normalized to the number ofcells analyzed. Information regarding DR5 can thus be used to aid indetermining stage or grade of a cancer by correlating the level of theDR5 protein (or fragment peptides of the DR5 protein) with levelsobserved in normal tissues.

Once the stage and/or grade, and/or DR5 protein expressioncharacteristics of the cancer has been determined, that information canbe matched to a list of therapeutic agents (chemical and biological)developed to specifically treat cancer tissue that is characterized by,for example, abnormal expression of the protein or protein(s) (e.g.,DR5) that were assayed. Matching information from an DR5 protein assayto a list of therapeutic agents that specifically targets, for example,the DR5 protein or cells/tissue expressing the protein, defines what hasbeen termed a personalized medicine approach to treating disease. Theassay methods described herein form the foundation of a personalizedmedicine approach by using analysis of proteins from the patient's owntissue as a source for diagnostic and treatment decisions. The DR5protein can be analyzed individually or in combination with otherproteins in a personalized medicine approach to diagnosing and treatingcancer.

These and other aspects of the present disclosure will become apparentto the skilled person in view of the description set forth below.

DETAILED DESCRIPTION

The Selected Reaction Monitoring/Multiple Reaction Monitoring (SRM/MRM)assay can be used to measure relative or absolute quantitative levels ofone or more of the specific peptides from the DR5 protein, individually,in combinations, or simultaneously, and therefore provide a means ofmeasuring the amount of the DR5 protein in a given protein preparationobtained from a biological sample by mass spectrometry.

More specifically, the SRM/MRM assay can measure these peptides directlyin complex protein lysate samples prepared from cells procured frompatient tissue samples, such as formalin fixed cancer patient tissue.Methods of preparing protein samples from formalin fixed tissue aredescribed in U.S. Pat. No. 7,473,532, the contents of which are herebyincorporated by references in their entireties. The methods described inU.S. Pat. No. 7,473,532 may conveniently be carried out using LiquidTissue™ reagents and protocol available from OncoPlexDx (formerlyExpression Pathology Inc., Rockville, Md.).

The most widely and advantageously available form of tissues from cancerpatient tissue is formalin fixed, paraffin embedded tissue.Formaldehyde/formalin fixation of surgically removed tissue is by farand away the most common method of preserving cancer tissue samplesworldwide and is the accepted convention for standard pathologypractice. Aqueous solutions of formaldehyde are referred to as formalin.“100%” formalin consists of a saturated solution of formaldehyde (thisis about 40% by volume or 37% by mass) in water, with a small amount ofstabilizer, usually methanol to limit oxidation and degree ofpolymerization. The most common way in which tissue is preserved is tosoak whole tissue for extended periods of time (8 hours to 48 hours) inaqueous formaldehyde, commonly termed 10% neutral buffered formalin,followed by embedding the fixed whole tissue in paraffin wax for longterm storage at room temperature. Thus molecular analytical methods toanalyze formalin fixed cancer tissue will be the most accepted andheavily utilized methods for analysis of cancer patient tissue.

In principle, any predicted peptide derived from the DR5 protein,prepared for example by digesting with a protease of known specificity(e.g. trypsin), can be used as a surrogate reporter to determine theabundance of DR5 protein in a sample using a mass spectrometry-basedSRM/MRM assay. Similarly, any predicted peptide sequence containing anamino acid residue at a site that is known to be potentially modified inthe DR5 protein also might potentially be used to assay the extent ofmodification of the DR5 protein in a sample.

According to one embodiment, DR5 fragment peptides may be generated in avariety of ways including using the Liquid Tissue™ protocol described inU.S. Pat. No. 7,473,532. The Liquid Tissue™ protocol and reagentsproduce peptide samples suitable for mass spectroscopic analysis fromformalin fixed paraffin embedded tissue by proteolytic digestion of theproteins in the tissue/biological sample. Suitable reagents andprotocols also are commercially available from OncoPlexDx (formerlyExpression Pathology Inc., Rockville, Md.).

In the Liquid Tissue™ protocol the tissue/biological sample is heated ina buffer for an extended period of time (e.g., from about 80° C. toabout 100° C. for a period of time from about 10 minutes to about 4hours) to reverse or release protein cross-linking. The buffer employedis a neutral buffer, (e.g., a Tris-based buffer, or a buffer containinga detergent). Following heat treatment the tissue/biological sample istreated with one or more proteases including, but not limited to,trypsin, chymotrypsin, pepsin, and endoproteinase Lys-C for a timesufficient to disrupt the tissue and cellular structure of saidbiological sample and to liquefy the sample. Exemplary conditions forthe protease treatment are from 30 minutes to 24 hours at a temperaturefrom 37° C. to 65° C.). Advantageously, endoproteases, and particularlycombinations of two or three endoproteases, used either simultaneouslyor sequentially, are employed to liquefy the sample. For example,suitable combinations of proteases can include, but are not limited to,combinations of trypsin, endoproteinase Lys-C and chemotrypsin, such astrypsin and endoproteinase Lys-C. The result of the heating andproteolysis is a liquid, soluble, dilutable biomolecule lysate.Advantageously, this liquid lysate is free of solid or particulatematter that can be separated from the lysate by centrifugation.

Surprisingly, it was found that many potential peptide sequences fromthe DR5 protein are unsuitable or ineffective for use in massspectrometry-based SRM/MRM assays for reasons that are not immediatelyevident. As it was not possible to predict the most suitable peptidesfor MRM/SRM assay, it was necessary to experimentally identify modifiedand unmodified peptides in actual Liquid Tissue™ lysates to develop areliable and accurate SRM/MRM assay for the DR5 protein. While notwishing to be bound by any theory, it is believed that some peptidesmight, for example, be difficult to detect by mass spectrometry becausethey do not ionize well or produce fragments that are not distinct fromthose generated from other proteins. Peptides may also fail to resolvewell in separation (e.g., liquid chromatography), or may adhere to glassor plastic ware, which leads to erroneous results in the SRM/MRM assay.

DR5 peptides found in various embodiments of this disclosure (forexample, Tables 1 and 2 below) were derived from the DR5 protein byprotease digestion of all the proteins within a complex Liquid Tissue™lysate prepared from cells procured from formalin fixed cancer tissue.Unless noted otherwise, in each instance the protease was trypsin. TheLiquid Tissue™ lysate was then analyzed by mass spectrometry todetermine those peptides derived from the DR5 protein that are detectedand analyzed by mass spectrometry. Identification of a specificpreferred subset of peptides for mass-spectrometric analysis is basedon: 1) experimental determination of which peptide or peptides from aprotein ionize in mass spectrometry analyses of Liquid Tissue™ lysates,and 2) the ability of the peptide to survive the protocol andexperimental conditions used in preparing a Liquid Tissue™ lysate. Thislatter property extends not only to the amino acid sequence of thepeptide but also to the ability of a modified amino acid residue withina peptide to survive in modified form during the sample preparation.

Protein lysates from cells procured directly from formalin(formaldehyde) fixed tissue were prepared using the Liquid Tissue™reagents and protocol. This entails collecting cells into a sample tubevia tissue microdissection followed by heating the cells in the LiquidTissue™ buffer for an extended period of time. Once the formalin-inducedcross linking has been negatively affected, the tissue/cells are thendigested to completion in a predictable manner using a protease such as,trypsin. The skilled artisan will recognize that other proteases, and inparticular, endoproteases may be used in place of, or in addition to,trypsin. Each protein lysate was used to prepare a collection ofpeptides by digestion of intact polypeptides with the protease orprotease combination. Each Liquid Tissue™ lysate was analyzed (e.g., byion trap mass spectrometry) to perform multiple global proteomic surveysof the peptides where the data was presented as identification of asmany peptides as could be identified by mass spectrometry from allcellular proteins present in each protein lysate. An ion trap massspectrometer or another form of a mass spectrometer that is capable ofperforming global profiling for identification of as many peptides aspossible from a single complex protein/peptide lysate may be employed.Ion trap mass spectrometers may, however, be the best type of massspectrometer for conducting global profiling of peptides. AlthoughSRM/MRM assays can be developed and performed on any type of massspectrometer, including a MALDI, ion trap, or triple quadrupole, aninstrument platform for SRM/MRM assay is often considered to be a triplequadrupole instrument platform.

Once as many peptides as possible were identified in a single massspectrometric analysis of a single lysate under the conditions employed,then the list of identified peptides was collated and used to determinethe proteins that were detected in that lysate. This process wasrepeated for multiple Liquid Tissue™ lysates, and the very large list ofpeptides was collated into a single dataset. The resulting datasetrepresents the peptides that can be detected in the type of biologicalsample that was analyzed (after protease digestion), and specifically ina Liquid Tissue™ lysate of the biological sample, and thus includes thepeptides for specific proteins, such as for example the DR5 protein.

In one embodiment, the DR5 tryptic peptides identified as useful in thedetermination of absolute or relative amounts of the DR5 protein includeone or more, two or more, or three of the peptides of SEQ ID NO:1, SEQID NO:2, and SEQ ID NO:3, each of which are shown in Table 1. Each ofthose peptides was detected by mass spectrometry in Liquid Tissue™lysates prepared from formalin fixed, paraffin embedded tissue. Thus,each of the peptides in Table 1, or any combination of those peptides(e.g., one or more, two or more, or three of those peptides recited inTable 1) are candidates for use in quantitative SRM/MRM assay for theDR5 protein in human biological samples, including directly in formalinfixed patient tissue. Table 2 shows additional information regarding thepeptides shown in Table 1.

TABLE 1 SEQ ID Peptide sequence SEQ ID NO: 1 LLVPANEGDPTETLRSEQ ID NO: 2 IEDHLLSSGK SEQ ID NO: 3 DASVHTLLDALETLGER

TABLE 2 Mono Precursor Precursor Transition Ion SEQ ID Peptide sequenceIsotopic Mass Charge State m/z m/z Type SEQ ID NO: 1 LLVPANEGDPTETLR1623.847 2 812.931 716.393 y6 2 812.931 888.442 y8 2 812.931 1017.484 y92 812.931 1131.527 y10 2 812.931 1299.617 y12 SEQ ID NO: 2 IEDHLLSSGK1097.572 2 549.793 491.282 y5 2 549.793 604.366 y6 2 549.793 741.425 y72 549.793 856.452 y8 SEQ ID NO: 3 DASVHTLLDALETLGER 1838.937 2920.4749756 888.478 y8 2 920.4749756 1003.505 y9 2 920.4749756 1116.589y10 2 920.4749756 1229.673 y11

The DR5 tryptic peptides listed in Table 1 include those detected frommultiple Liquid Tissue™ lysates of multiple different formalin fixedtissues of different human organs including prostate, colon, and breast.Each of those peptides is considered useful for quantitative SRM/MRMassay of the DR5 protein in formalin fixed tissue. Further data analysisof these experiments indicated no preference is observed for anyspecific peptides from any specific organ site. Thus, each of thesepeptides is believed to be suitable for conducting SRM/MRM assays of theDR5 protein on a Liquid Tissue™ lysate from any formalin fixed tissueoriginating from any biological sample or from any organ site in thebody.

One consideration for conducting an SRM/MRM assay is the type ofinstrument that may be employed in the analysis of the peptides.Although SRM/MRM assays can be developed and performed on any type ofmass spectrometer, including a MALDI, ion trap, or triple quadrupole,the most advantageous instrument platform for SRM/MRM assay is oftenconsidered to be a triple quadrupole instrument platform. That type of amass spectrometer may be considered to be the most suitable instrumentfor analyzing a single isolated target peptide within a very complexprotein lysate that may consist of hundreds of thousands to millions ofindividual peptides from all the proteins contained within a cell.

In order to most efficiently implement an SRM/MRM assay for each peptidederived from the DR5 protein it is desirable to utilize information inaddition to the peptide sequence in the analysis. That additionalinformation may be used in directing and instructing the massspectrometer (e.g. a triple quadrupole mass spectrometer), to performthe correct and focused analysis of specific targeted peptide(s), suchthat the assay may be effectively performed.

The additional information about target peptides in general, and aboutspecific DR5 peptides, may include one or more of the mono isotopic massof the peptide, its precursor charge state, the precursor m/z value, them/z transition ions, and the ion type of each transition ion. Additionalpeptide information that may be used to develop an SRM/MRM assay for theDR5 protein is shown in Table 2.

The methods described below can be used to: 1) identify candidatepeptides from the DR5 protein that can be used for a massspectrometry-based SRM/MRM assay for the DR5 protein, 2) developindividual SRM/MRM assays, or multiplexed assays, for target peptidesfrom the DR5 protein in order to correlate to cancer, and 3) applyquantitative DR5 assays to cancer diagnosis and/or choice of optimaltherapy for cancer.

Assay Methods

I. Identification of SRM/MRM Candidate Fragment Peptides for the DR5Protein:

-   -   a. Prepare a Liquid Tissue™ protein lysate from a formalin fixed        biological sample using a protease or proteases, (that may or        may not include trypsin), to digest proteins    -   b. Analyze all protein fragments in the Liquid Tissue™ lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the DR5 protein, where individual fragment        peptides do not contain any peptide modifications such as        phosphorylations or glycosylations    -   c. Analyze all protein fragments in the Liquid Tissue™ lysate on        an ion trap tandem mass spectrometer and identify all fragment        peptides from the DR5 protein that carry peptide modifications        such as for example phosphorylated or glycosylated residues    -   d. All peptides generated by a specific digestion method from        the entire, full length DR5 protein potentially can be measured,        but preferred peptides used for development of the SRM/MRM assay        are those that are identified by mass spectrometry directly in a        complex Liquid Tissue™ protein lysate prepared from a formalin        fixed biological sample    -   e. Peptides that are specifically modified (phosphorylated,        glycosylated, etc.) in patient tissue and which ionize, and thus        detected, in a mass spectrometer when analyzing a Liquid Tissue™        lysate from a formalin fixed biological sample are identified as        candidate peptides for assaying peptide modifications of the DR5        protein        II. Mass Spectrometry Assay for Fragment Peptides from DR5        Protein    -   a. SRM/MRM assay on a triple quadrupole mass spectrometer for        individual fragment peptides identified in a Liquid Tissue™        lysate is applied to peptides from the DR5 protein        -   i. Determine optimal retention time for a fragment peptide            for optimal chromatography conditions including but not            limited to gel electrophoresis, liquid chromatography,            capillary electrophoresis, nano-reversed phase liquid            chromatography, high performance liquid chromatography, or            reverse phase high performance liquid chromatography        -   ii. Determine the mono isotopic mass of the peptide, the            precursor charge state for each peptide, the precursor m/z            value for each peptide, the m/z transition ions for each            peptide, and the ion type of each transition ion for each            fragment peptide in order to develop an SRM/MRM assay for            each peptide.        -   iii. SRM/MRM assay can then be conducted using the            information from (i) and (ii) on a triple quadrupole mass            spectrometer where each peptide has a characteristic and            unique SRM/MRM signature peak that precisely defines the            unique SRM/MRM assay as performed on a triple quadrupole            mass spectrometer    -   b. Perform SRM/MRM analysis so that the amount of the fragment        peptide of the DR5 protein that is detected, as a function of        the unique SRM/MRM signature peak area from an SRM/MRM mass        spectrometry analysis, can indicate both the relative and        absolute amount of the DR5 protein in a particular protein        lysate.        -   i. Relative quantitation may be achieved by:            -   1. Determining increased or decreased presence of the                DR5 protein by comparing the SRM/MRM signature peak area                from a given DR5 peptide detected in a Liquid Tissue™                lysate from one formalin fixed biological sample to the                same SRM/MRM signature peak area of the same DR5                fragment peptide in at least a second, third, fourth or                more Liquid Tissue™ lysates from least a second, third,                fourth or more formalin fixed biological samples.            -   2. Determining increased or decreased presence of the                DR5 protein by comparing the SRM/MRM signature peak area                from a given DR5 peptide detected in a Liquid Tissue™                lysate from one formalin fixed biological sample to                SRM/MRM signature peak areas developed from fragment                peptides from other proteins, in other samples derived                from different and separate biological sources, where                the SRM/MRM signature peak area comparison between the 2                samples for a peptide fragment are normalized to amount                of protein analyzed in each sample.            -   3. Determining increased or decreased presence of the                DR5 protein by comparing the SRM/MRM signature peak area                for a given DR5 peptide to the SRM/MRM signature peak                areas from other fragment peptides derived from                different proteins within the same Liquid Tissue™ lysate                from the formalin fixed biological sample in order to                normalize changing levels of DR5 protein to levels of                other proteins that do not change their levels of                expression under various cellular conditions.            -   4. These assays can be applied to both unmodified                fragment peptides and for modified fragment peptides of                the DR5 protein, where the modifications include but are                not limited to phosphorylation and/or glycosylation, and                where the relative levels of modified peptides are                determined in the same manner as determining relative                amounts of unmodified peptides.        -   ii. Absolute quantitation of a given peptide may be achieved            by comparing the SRM/MRM signature peak area for a given            fragment peptide from the DR5 protein in an individual            biological sample to the SRM/MRM signature peak area of an            internal fragment peptide standard spiked into the protein            lysate from the biological sample.            -   1. The internal standard is a labeled synthetic version                of the fragment peptide from the DR5 protein that is                being interrogated. This standard is spiked into a                sample in known amounts, and the SRM/MRM signature peak                area can be determined for both the internal fragment                peptide standard and the native fragment peptide in the                biological sample separately, followed by comparison of                both peak areas.            -   2. This can be applied to unmodified fragment peptides                and modified fragment peptides, where the modifications                include but are not limited to phosphorylation and/or                glycosylation, and where the absolute levels of modified                peptides can be determined in the same manner as                determining absolute levels of unmodified peptides.                III. Apply Fragment Peptide Quantitation to Cancer                Diagnosis and Treatment    -   a. Perform relative and/or absolute quantitation of fragment        peptide levels of the DR5 protein and demonstrate that the        previously-determined association, as well understood in the        field of cancer, of DR5 protein expression to the        stage/grade/status of cancer in patient tumor tissue is        confirmed.    -   b. Perform relative and/or absolute quantitation of fragment        peptide levels of the DR5 protein individually, in combinations,        or all simultaneously, and demonstrate correlation with clinical        outcomes from different treatment strategies, wherein this        correlation has already been demonstrated in the field or can be        demonstrated in the future through correlation studies across        cohorts of patients and tissue from those patients. Once either        previously established correlations or correlations derived in        the future are confirmed by this assay then the assay method can        be used to determine optimal treatment strategy.

The information shown in Table 2 is necessary to develop an SRM/MRMassay for quantitation of the DR5 protein on a triplequadrupole massspectrometer. Specific and unique characteristics about these DR5peptides were developed by analysis of all DR5 peptides on an ion trapand/or triple quadrupole mass spectrometers. That information includesthe monoisotopic mass of the peptide, its precursor charge state, theprecursor m/z value, the transition m/z values of the precursor, and theion types of each of the identified transitions. That information mustbe determined experimentally for each and every candidate SRM/MRMpeptide directly in Liquid Tissue™ lysates from formalin fixed tissue;because, interestingly, not all peptides from the DR5 protein can bedetected in such lysates using SRM/MRM as described herein, indicatingthat DR5 peptides not detected cannot be considered candidate peptidesfor developing an SRM/MRM assay for use in quantitatingpeptides/proteins directly in Liquid Tissue™ lysates from formalin fixedtissue.

Utilizing this information, quantitative SRM/MRM assays can be developedfor the DR5 protein, and assessment of DR5 protein levels in tissuesbased on analysis of formalin fixed patient-derived tumor tissue canprovide diagnostic, prognostic, and therapeutically-relevant informationabout each particular cancer patient.

In one embodiment, this disclosure provides methods for measuring thelevel of the DR5 protein in a biological sample, comprising detectingand/or quantifying the amount of one or more modified or unmodified DR5fragment peptides in a protein digest prepared from the biologicalsample using mass spectrometry; and calculating the level of modified orunmodified DR5 protein in the sample; and wherein the level is arelative level or an absolute level. In another embodiment, thisdisclosure provides a method for quantifying one or more DR5 fragmentpeptides, wherein the method comprises determining the amount of one ormore of the DR5 fragment peptides in a biological sample by comparisonto an added internal standard peptide of known amount, wherein each ofthe DR5 fragment peptides in the biological sample is compared to aninternal standard peptide having the same amino acid sequence. In someembodiments the internal standard is an isotopically labeled internalstandard peptide comprising one or more heavy stable isotopes selectedfrom ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, ²H or combinations thereof.

The methods for measuring levels of the DR5 protein in a biologicalsample described herein (or fragment peptides as surrogates thereof) areuseful as diagnostic indicators of cancer in a patient or subject. Inone embodiment, the results from the measurements of levels of the DR5protein may be employed to determine the diagnostic stage/grade/statusof a cancer by correlating (e.g., comparing) the level of DR5 proteinfound in a tissue with the level of these proteins found in normaland/or cancerous or precancerous tissues.

Embodiments

1. A method for measuring the amount of the DR5 protein in a biologicalsample, comprising detecting and/or quantifying the level of one or moremodified or unmodified DR5 fragment peptides in a protein digestprepared from the biological sample using mass spectrometry; andcalculating the level of modified or unmodified DR5 protein in thesample; and

-   -   wherein the amount is a relative amount or an absolute amount.

2. The method of embodiment 0, further comprising the step offractionating the protein digest prior to detecting and/or quantifyingthe level of one or more modified or unmodified DR5 fragment peptides.

3. The method of embodiment 2, wherein the fractionating step isselected from the group consisting of gel electrophoresis, liquidchromatography, capillary electrophoresis, nano-reversed phase liquidchromatography, high performance liquid chromatography, and reversephase high performance liquid chromatography.

4. The method of any of embodiments 1 to 3, wherein the protein digestof the biological sample is prepared by the Liquid Tissue™ protocol.

5. The method of any of embodiments 1 to 3, wherein the protein digestcomprises a protease digest.

6. The method of embodiment 5, wherein the protein digest comprises atrypsin digest.

7. The method of any of embodiments 1 to 6, wherein the massspectrometry comprises tandem mass spectrometry, ion trap massspectrometry, triple quadrupole mass spectrometry, MALDI-TOF massspectrometry, MALDI mass spectrometry, or time of flight massspectrometry, or any combination thereof.

8. The method of embodiment 7, wherein the mode of mass spectrometryused is Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring(MRM), or multiple Selected Reaction Monitoring (mSRM), or anycombination thereof.

9. The method of any of embodiments 1 to 8, wherein the one or moremodified or unmodified DR5 fragment peptides comprise different aminoacid sequences independently selected from those set forth as SEQ IDNO:1, SEQ ID NO:2, and SEQ ID NO:3.

10. The method of any of embodiments 0 to 9, wherein the biologicalsample is a blood sample, a urine sample, a serum sample, an ascitessample, a sputum sample, lymphatic fluid, a saliva sample, a cell, or asolid tissue.

11. The method of embodiment 10, wherein the tissue is formalin fixedtissue.

12. The method of embodiment 10 or 11, wherein the tissue is paraffinembedded tissue.

13. The method of embodiment 10, wherein the tissue is obtained from atumor.

14. The method of embodiment 13, wherein the tumor is a primary tumor.

15. The method of embodiment 13, wherein the tumor is a secondary tumor.

16. The method of any of embodiments 0 to 15, further comprisingquantifying a modified or unmodified DR5 fragment peptide.

17. The method of embodiment 16, wherein quantifying a modified orunmodified fragment peptide comprises comparing the amount of one ormore DR5 fragment peptides comprising an amino acid sequence of about 8to about 45 amino acid residues of DR5 as shown in SEQ ID NO:1, SEQ IDNO:2, and SEQ ID NO:3 in one biological sample to the amount of the sameDR5 fragment peptide in a different and separate biological sample.

18. The method of embodiment 17, wherein quantifying one or moremodified or unmodified DR5 fragment peptides comprises determining theamount of the each of said one or more DR5 fragment peptides in abiological sample by comparison to an added internal standard peptide ofa known amount, wherein each of the DR5 fragment peptides in thebiological sample is compared to an internal standard peptide having thesame amino acid sequence.

19. The method of embodiment 18, wherein the internal standard peptideis an isotopically labeled peptide.

20. The method of embodiment 19, wherein the isotopically labeledinternal standard peptide comprises one or more heavy stable isotopesselected from the group consisting of ¹⁸O, ¹⁷O, ³⁴S, ¹⁵N, ¹³C, and ²H,or any combinations thereof.

21. The method of any of embodiments 1 to 20, wherein detecting and/orquantifying the amount of one or more modified or unmodified DR5fragment peptides in the protein digest indicates the presence ofmodified or unmodified DR5 protein and an association with cancer in thesubject.

22. The method of embodiment 21, further comprising correlating theresults of detecting and/or quantifying amounts of one or more modifiedor unmodified DR5 fragment peptides, or the amount of the DR5 protein tothe diagnostic stage/grade/status of the cancer.

23. The method of embodiment 22, wherein correlating the results ofdetecting and/or quantifying the amount of one or more modified orunmodified DR5 fragment peptides, or the amount of the DR5 protein tothe diagnostic stage/grade/status of the cancer is combined withdetected and/or quantified amounts of other proteins, or peptides fromother proteins, in a multiplex format to provide additional informationabout the diagnostic stage/grade/status of the cancer.

24. The method of any one of embodiments 1 to 23, further comprisingselecting for the subject, from which the biological sample is obtained,a treatment based on the presence, absence, or amount of one or more DR5fragment peptides or the amount of DR5 protein.

25. The method of any one of embodiments 1 to 24, further comprisingadministering to the patient from which the biological sample isobtained a therapeutically effective amount of a therapeutic agent,wherein the therapeutic agent and/or amount of the therapeutic agentadministered is based upon the amount of one or more modified orunmodified DR5 fragment peptides or the amount of DR5 protein.

26. The method of embodiments 24 or 25, wherein the treatment or thetherapeutic agent is directed to cancer cells expressing DR5 protein.

27. The method of any of embodiments 1 to 26, wherein the biologicalsample is formalin fixed tumor tissue that has been processed forquantifying the amount of one or more modified or unmodified DR5fragment peptides employing the Liquid Tissue™ protocol and reagents.

28. The method of any of embodiments 1-27, wherein said one or moremodified or unmodified DR5 fragment peptides is one, two, or more of thepeptides in Table 1.

29. The method of any of embodiments 1-28, comprising quantifying theamount of the peptides in Table 2.

30. A composition comprising one, two, or three of the peptides in Table1 or antibodies thereto, said composition optionally excluding one, two,three or more peptides of DR5 that are not peptides of SEQ ID NOs: 1, 2,and/or 3.

31. The composition of embodiment 30, comprising two, or three of thepeptides of Table 2 or antibodies thereto.

It is to be understood that the description, specific examples,embodiments, and data, while indicating exemplary aspects, are given byway of illustration and are not intended to limit the resent disclosure.Various changes and modifications within the present disclosure willbecome apparent to the skilled artisan from the discussion, detaileddescription, and data contained herein, and thus are considered part ofthe subject matter of this application optionally excluding one, two,three or more peptides of DR5 that are not peptides of SEQ ID NOs: 1, 2,and/or 3.

The invention claimed is:
 1. A method for measuring the amount of theDeath Receptor 5 (DR5) protein in a human biological sample offormalin-fixed tissue, comprising detecting and quantifying the amountof a DR5 fragment peptide in a protein digest prepared from said humanbiological sample using mass spectrometry, wherein the DR5 fragmentpeptide has the sequence of SEQ ID NO:1; and calculating the level ofmodified or unmodified DR5 protein in the sample; wherein the amount isa relative amount or an absolute amount, wherein detecting andquantifying the amount of said DR5 fragment peptide in said proteindigest indicates the presence of DR5 protein and an association withcancer in the subject.
 2. The method of claim 1, further comprisingcorrelating the results of detecting and quantifying the amount of saidDR5 fragment peptide, or the amount of the DR5 protein to the diagnosticstage/grade/status of the cancer.
 3. The method of claim 2, whereincorrelating the results of detecting and quantifying the amount of saidDR5 fragment peptide, or the amount of the DR5 protein to the diagnosticstage/grade/status of the cancer is combined with detected and/orquantified amounts of other proteins, or peptides from other proteins,in a multiplex format to provide additional information about thediagnostic stage/grade/status of the cancer.